1. Field of the Invention
The invention involves a novel method and apparatus for obtaining pure cell populations or cell constituents such as DNA, RNA or proteins from target cells in tissue sections using ultraviolet (UV) laser-assisted ablation of non-target cells.
2. Description of the Background Art
Cancer is a leading cause of death in the United States. Treatments for cancers include surgery, chemotherapy, and radiation therapy, which cause considerable morbidity and often are ineffective. Standard pathological grading and staging cannot predict the susceptibility of a particular tumor to eradication by chemotherapy, radiation therapy, or other therapy, and thus many patients with solid tumors receive ineffective toxic therapy. Better prognostic indicators and therapeutic targets are needed for cancer treatment.
A large worldwide effort is underway to develop improved prognostic and therapeutic tools for cancer. New molecular biology techniques permit investigation of specific genetic alterations in cancers. Evidence is accumulating that information about specific DNA alterations in tumors, which predict cancer behavior, may provide important new tools for cancer diagnosis, prognosis, and therapy.
For example, a recent study found that in one of the most common cancers in children (Wilms"" tumor), tumor-specific loss of heterozygosity of chromosome 16q predicts adverse outcome independent of histological type. Based on this knowledge, the subgroup of Wilms"" tumor patients with favorable histology and loss of heterozygosity for chromosome 16q in their tumors may now benefit from earlier, more aggressive therapy. Further, in colon cancer, the status of chromosome 18q has recently been shown to have strong prognostic value in patients with cancer extending through the bowel without lymph node metastasis (stage II). Thus, stage II colon cancer patients with tumor specific loss of heterozygosity on chromosome 18q are a newly defined subset of patients that may benefit from more aggressive adjuvant therapy at the time of their initial diagnosis.
Other types of tumor-specific genetic alterations, including amplification of specific alleles and inactivation of specific genes or alleles by cytidine methylation have shown promise for providing important new prognostic information. It may be possible to define the xe2x80x9csignaturexe2x80x9d of genetic lesions in an individual patient""s tumor, permitting therapy tailored specifically to the genetic defects of the tumor.
Current molecular biologic techniques allow the study of DNA, RNA, and protein contained within cells. Some techniques allow cells to be studied in situ, with labelled molecular probes visualized under a microscope. These techniques are very useful, but currently are quite limited in their resolution and consistency. Other more powerful techniques for studying cellular DNA, RNA or protein depend on pooling of material from one or several cells. Studies based on such pooling have identified the first known gene-specific changes associated with cancer and other diseases, and have provided insight into the molecular processes involved in the transformation of cells from normal to abnormal.
Understanding molecular genetic changes involved in the pathogenesis of organ dysfunction requires studying groups of diseased cells in isolation and comparing them to phenotypically normal cells. The difficulty is that diseased cells in any tissue are usually accompanied by many phenotypically normal cells. Thus molecular studies reported to date have been limited to tissues in which the concentration of diseased cells is relatively high, such as in large, concentrated tumor masses. Various researchers have attempted to obtain purer samples of DNA, RNA, or protein from diseased cells by scraping portions of tissue sections away with a cutting instrument or by inking target areas of tissue sections and later exposing the section to UV light to destroy non-inked DNA and RNA.
Ultraviolet irradiation of such tissue has been found to cause single and double stranded DNA breaks, DNA crosslinks, generation of local denatured sites in DNA and DNA base destruction. Thus, it is known that ultraviolet irradiation of a tissue section can massively disrupt the DNA strand (as well as RNA and protein) contained within that tissue section. For example, selective UV irradiation (non-laser) exposure of portions of tissue sections was achieved by Shibata et al. (Amer. J. Pathol. 141(3):539-543, 1992) by covering target areas of stained tissue sections with black ink and UV irradiating the entire tissue sample with a standard broad spectrum UV light bulb. Shibata demonstrated that DNA within cells covered with the black ink is preserved, while DNA in UV exposed adjacent portions of the tissue was destroyed. This crude technique is markedly limited by the width of the black marking pen used, by difficulty in directing the pen to the desired location, and by the need to continually replace the pen in order to avoid contamination of inked areas by cellular material from areas previously inked. Another limitation is that inking must be performed while no cover slip is in place, markedly reducing optical resolution and making visual identification of cells nearly impossible in many cases.
Formalin fixed, paraffin embedded (FFPE) tissues are the basis for current pathology practice. They are readily available to most pathologists and cancer researchers and provide histological detail that remains the benchmark for pathology. FFPE tissue is not ideal for many molecular methods because DNA and RNA contained within this tissue is partially degraded. Although it is more difficult to isolate DNA of adequate quality for analysis from FFPE tissue sections than from unfixed, unembedded tissue, a number of studies have demonstrated that amplification of DNA fragments as long as 536 base pairs can be accomplished with tissue fixed in buffered formalin. However, the duration of storage, fixative used, fixation time, fixation temperature, and extraction procedures all affect the quality of DNA that can be isolated from paraffin. Recent molecular techniques have allowed a wide range of genetic alterations to be detected in DNA and RNA isolated from archival tissues. Most of these techniques are based on Polymerase Chain Reaction (xe2x80x9cPCRxe2x80x9d).
PCR based genetic analysis of single cells or groups of cells has been used to discover molecular alterations in cells. For example, PCR techniques have been used to detect loss of heterozygosity, genomic DNA mutation, mitochondrial DNA mutation, DNA methylation, gene dosage, gene rearrangements, clonality and detection of DNA adducts. However, because cancer cells grow in close relation to noncancerous cells in all tissues, it is nearly impossible using heretofore known techniques to obtain pure tumor DNA. Hence, background signals from noncancerous cells often distort the analysis of genetic changes in tumors. For example, when a mutation is not detected in a particular gene in DNA isolated from a tumor, it is quite possible that the nonmutated sequence came from noncancerous cells"" DNA contaminating the sample. This contamination problem was demonstrated in a controversy concerning the importance of the recently identified gene p16/MTS1. One of the gene""s discoverers cast doubt on analyses of certain DNA samples which did not show p16 mutations because of contamination by noncancerous DNA.
The problem of background noise created by contaminating noncancerous cells was again emphasized by difficulty in identifying mutations in the breast cancer associated gene BRCA1 in sporadic and hereditary tumors. In cases of hereditary tumors, the individual inherits one mutated copy of the gene. Researchers have had difficulty studying, the remaining copy of the gene in hereditary tumor samples because of background noise from contaminating normal cells, making it difficult to ascertain the frequency of specific BRCA1 genetic alterations. Because of the difficulty of obtaining pure breast cancer DNA samples in general, it is not known with confidence that in fact BRCA1 mutations are rare in non-hereditary breast tumors. Interpretation of results based on impure tissue samples are ambiguous in direct proportion to the degree of impurity of the DNA, RNA or protein isolated.
Current techniques of DNA, RNA and protein isolation and purification illustrate the problem of sample purity in researching genetic alterations in cancer.
For example, serial cryostat sectioning and trimming of frozen tumor-bearing tissue has been used to produce limited purification of tumor DNA and has been useful in the definition of loss of heterozygosity and other genetic alterations. Unfortunately, this cryostat-based xe2x80x9cfractionationxe2x80x9d of cancer tissue has a number of drawbacks. For example, most common primary tumors, including breast, ovarian, pancreatic, prostate cancers and others, are highly infiltrated with noncancerous cells. Hence, it is difficult to obtain better than 70% purity in the majority of cases using cryostat sectioning alone, since only relatively large regions of the tissue can be carved manually from the tissue block. Further, histological detail is often poor in frozen sectioned material, making interpretation difficult. In addition, cryostat methods cannot be applied easily to the clinical setting because clinicians find it inconvenient to freeze biopsy samples, and pathologists prefer paraffin embedded material for interpretation.
Improved signal (cancerous DNA) to noise (noncancerous DNA) ratio in PCR based allelic loss analysis has been achieved by mechanical microdissection of individual tissue sections or by a combination of broadband ultraviolet light treatment of the section after covering desired areas with black ink, followed by mechanical removal of the tissue from the slide. However, these manual microdissection methods have several drawbacks. For example, microdissection or ink-dotting must be performed without a cover slip in place which markedly reduces optical resolution. Further, manual dissection or ink dotting only allows the selective isolation of clumps of cells.
Microdissection using stage-mounted micromanipulators has been reported, but the lack of resolution without a coverslip in place typically precludes isolation of single cells. Moreover, the microdissection and ink-dotting techniques are extremely time-consuming and cumbersome.
In situ hybridization is another method of locating specific base sequences in tissue sections. For example, fluorescence in situ hybridization (FISH) has been used to show sequence deletions and ploidy anomalies in prostate tumors. However, in situ methods have several disadvantages. For example, genetic material in the section is consumed by the process and is not available for multiple analyses. In addition, the specificity of individual FISH probes is highly variable. Finally, many molecular alterations (mutations, methylation, small deletions, etc.) are not detectable with FISH.
Non-histological methods for enrichment of tissue samples of tumor cells are also possible, such as separation of ovarian tumor cells using flow cytometry, and enrichment of breast and prostate tumor cells with an avidin affinity column. However, these techniques are significantly limited because they require a tumor specific antibody, which is not available for the majority of tumors. Moreover, even if tumor specific antibodies were available for all tumors, use in a clinical setting would require separate separation protocols and columns for every tumor type, which would be very inefficient. Furthermore, specific antibodies are notoriously variable in their staining characteristics. Finally, these xe2x80x9cblindxe2x80x9d separation methods make it difficult to obtain both histological grading and molecular diagnostic information on the same tissue sample. Histological grading of tumors is one of the most powerful prognostic tools available and any new molecular analysis will be compared to standard histological grading techniques to determine their prognostic value, a comparison that will not easily be achieved with flow cytometry based or affinity column based purification systems. A method that combines the ability to obtain maximum enrichment of tumor cells while maintaining histological analysis of the sample has significant advantages over flow cytometry or column-based separation methods.
U.S. Pat. No. 5,272,081 discloses a method for selecting and separating individual cells in a sample for diagnostic purposes. In accordance with this method, cells are first separated, trapped in sized holes on a grid, each with a known location, and subjected to tests for selection. Once the cells on the grid have been tested and selected, the desired cells may be removed from the grid by selectively changing the electrical potential of conductors on the grid, or the undesired cells may be killed to effect separation. A major disadvantage of this method is that the cells must be suspended before they are trapped on the grid for testing. Cells in a standard paraffin embedded tissue sample, such as a biopsy sample, cannot be readily kept intact and separated by this method. Again, standard histological analysis of the same sample would be impossible, and usually the entire sample would be consumed by one experiment.
Also, U.S. Pat. Nos. 4,624,915 and 4,626,687 disclose methods and devices for the separation and segregation of living cells using a focused radiant energy beam. In accordance with these methods, living, anchorage-dependent target cells are moved on a microscope stage in two dimensions while the laser beam is directed through the objective lens of the microscope at individual living cells, or at the film supporting the cells. The living cells are viewed on the slides with no cover slip and while nutritive cell medium is flowing over the cells. Selection is based on a cellular light response to an attenuated laser beam, and undesired cells are killed with radiant energy from a focused, high power laser beam. Living cell selection can be accomplished by computer analysis of the fluorescence pattern of the living cells.
Freeze-dried tissue sections have been microdissected using a laser after first storing coordinates to be dissected from a non-freeze dried adjacent section using a drawing tube (Klimek et al., Carcinogenesis: 5 (2): 265-268, 1984). Because the tissue sections to be microdissected must be freeze-dried at xe2x88x9240xc2x0 C. and stored under vacuum at xe2x88x9245xc2x0 C., because the tissue shrinks when it is freeze dried, and because the regions to be dissected must be indirectly identified through the use of a drawing tube, this method is also very cumbersome and limited in precision. It does not allow specific individual cellular localization and ablation.
None of the known techniques contemplated for use with biopsy tissue samples have been used to reliably examine and selectively destroy individual cells, individual cell nuclei, or DNA within individual cells. The known techniques make it nearly impossible to routinely study small tissue samples in which there are a large percentage of non-diseased cells that cannot be cut out or selectively inked. Prostate biopsies, for example, may contain small foci of adenocarcinoma measuring 1 mm in diameter or less, surrounded by lymphocytes, smooth muscle and benign prostate cells. In most cases, it is extremely tedious or impossible to selectively destroy the lymphocytes, smooth muscle cells, and benign prostate cells using available techniques. These limitations make it difficult to bring routine pathologic diagnosis to the molecular level, because most tissue biopsies cannot be effectively studied using currently available techniques.
Using current tumor DNA purification techniques, noise from contaminating noncancerous DNA makes interpretation of data difficult and sometimes impossible, confounding cancer research and diagnosis. Accordingly, there is a profound need for a reliable technique to obtain highly enriched samples of tumor DNA, RNA and protein. Such a technique would be widely used by pathologists in clinical practice, cancer researchers, and in fact, any investigator who needed to purify individual cells or groups of cells from a heterogeneous tissue, cancerous or noncancerous.
In accordance with the present invention, a novel laser purification method and apparatus is disclosed for obtaining pure cell populations from a tissue section having a heterogenous cell population. This method allows one to obtain a tissue section having a heterogenous cell population which, after treatment, has relatively intact DNA, RNA or protein from only untreated cells. The invention comprises a microscope, a support stage for supporting the tissue section and a drive means for providing relative movement between the stage and the microscope in an x-y coordinate plane. A video camera is provided for scanning the tissue section and creating a mosaic of video images representing the tissue section. These high resolution video images are displayed on a video display. Target cells in the tissue section are microscopically identified using established morphological criteria or fluorescent tagged antibodies. In one embodiment, the operator inspects the video images representing the magnified tissue section displayed on the video display and manually selects the target cells to be destroyed or preserved utilizing a touch or light sensitive video display screen or cursor system. In another embodiment, the target cells of interest are automatically identified based upon an analysis of the video images by a computer. In this embodiment, the operator can verify the target cells identified by the computer and has the option of either selecting additional cells of interest or deselecting cells identified by the computer. After the manual or automatic identification and selection of the target cells, the computer automatically directs a laser to photoablate the DNA, RNA or protein in the contaminating cells so that the proportion of intact DNA and RNA from the target cells is markedly increased.
Further purification of the tissue section is achieved with the present invention by analyzing the cells not photoablated in the purification steps described above under a higher magnification, and repeating the photoablation process of the present invention. Purification, in accordance with one aspect of the present invention, means that the tissue section having a heterogeneous cell population, after treatment, will contain relatively intact DNA, RNA or protein from only the untreated cells.
The present invention is automated by using image analysis hardware and software, with minimal operator intervention, thereby allowing the DNA or RNA in selected portions of the tissue section to be automatically ablated, while the nonselected cells"" DNA or RNA will remain intact for subsequent analysis by PCR or other genetic methods.
Further, a greater degree of optical resolution is obtainable with the present invention over prior art systems since a UV transparent quartz cover slip can be used during operation of the viewing and laser devices.